Princeton University scientists report the construction of artificial proteins that enable the growth of living cells. The proteins were created by genetic sequences never before seen in nature and were shown to produce substances that sustain life in cells almost as readily as natural proteins.
“What we have here are molecular machines that function quite well within a living organism even though they were designed from scratch and expressed from artificial genes,” says Michael Hecht, Ph.D., a professor of chemistry at Princeton, who led the research. “This tells us that the molecular parts kit for life need not be limited to parts like genes and proteins that already exist in nature.”
The research appeared online January 4 in the journal Public Library of Science ONE in a paper titled “De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth.”
Most prior work in synthetic biology has focused on reorganizing parts drawn from natural organisms. In May 2010, scientists at the J. Craig Venter Institute (JCVI) created the first self-replicating bacterial cell that is driven by a synthetic, computer-designed genome constructed in the laboratory, completely from scratch. The team chemically synthesized small pieces of the 1.08 million base pair chromosome of a modified Mycoplasma mycoides genome, stitched the pieces together, and inserted the full length DNA into a Mycoplasma capricolum recipient cell.
Dr. Hecht notes that his team showed that biological functions can be provided by macromolecules that were not borrowed from nature but designed in the laboratory. He and his students were trying to figure out what processes drive the routine folding of proteins on a basic level and why certain key sequences have evolved to be central to existence. They questioned why there are only about 100,000 different proteins produced in the human body, when there is a potential for so many more. Were these particular proteins somehow special, or might others work equally well even though evolution has not yet had a chance to sample them?
Dr. Hecht and his research group thus set about to create artificial proteins encoded by genetic sequences not seen in nature. They produced about 1 million amino acid sequences that were designed to fold into stable 3-D structures. The collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles.
They probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of E. coli, each deleted for a conditionally essential gene. Four different strains were rescued by specific sequences from the library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ~0.1% of the E. coli genome (and ~1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.
This was significant because formation of a bacterial colony under these selective conditions could occur only if a protein in the collection had the capacity to sustain the growth of living cells.
“What I believe is most intriguing about our work is that the information encoded in these artificial genes is completely novel—it does not come from, nor is it significantly related to, information encoded by natural genes, and yet the end result is a living, functional microbe,” remarks Michael Fisher, co-author of the paper who earned his Ph.D. at Princeton in 2010 and is now a postdoctoral fellow at the University of California-Berkeley.
“It is perhaps analogous to taking a sentence, coming up with brand new words, testing if any of our new words can take the place of any of the original words in the sentence, and finding that in some cases, the sentence retains virtually the same meaning while incorporating brand new words.”
Kara McKinley, also a co-author and a 2010 Princeton graduate who is now a Ph.D. student at the Massachusetts Institute of Technology, comments, “This is an exciting result because it shows that unnatural proteins can sustain a natural system and that such proteins can be found at relatively high frequency in a library designed only for structure.”